Isolation of proteins involved in posttranscriptional gene silencing and methods of use

ABSTRACT

The present invention includes a method for detecting and isolating sugarcane proteins that interact with the HC-Pro and P1 proteins of SrMV and other proteins involved in gene silencing, particularly in sugarcane. The method uses a two hybrid assay with an HC-Pro, P1, or other silencing-related protein-containing bait protein and a prey protein containing a polypeptide encoded by a DNA molecule in a cDNA library. The method also includes identification of false positives through reverse two-hybrid assays and using in vitro techniques such as farwestern blots or pull down assays where plant physiological conditions may be replicated. Finally, interactions may be confirmed in planta. Some novel proteins used in and discovered using the these methods are also identified. Methods of using viral and plant proteins to regulate silencing in plants such as sugarcane are also discussed.

CLAIM TO PRIOR APPLICATIONS

[0001] The present application claims priority to U.S. ProvisionalPatent Application Ser. No. 60/314,863 filed on Aug. 24, 2001 andincorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates generally to sugarcane proteinisolation and more particularly to isolation and characterization ofproteins that are involved in posttranscriptional gene silencing. Theinvention also includes sugarcane and sorghum mosaic virus proteinsinvolved in silencing and the cDNAs which encode them. Finally theinvention includes use of these cDNAs and proteins to regulatesilencing.

BACKGROUND OF THE INVENTION

[0003] In the course of evolution organisms have developed a spectrum ofdefense mechanisms that target alien, parasitic elements. The mostspecialized defense system is perhaps the vertebrate immune system,which provides effective protection against a wide range of infectiousmicrobes. In the vertebrate immune system, it is essentially peptidesthat are recognized as non-self and eliminated. Transgenic plant studieshave revealed the existence of another, more ancestral level of defenseresponse. Homology-dependent gene silencing can be viewed as a novel,innate host defense system that is capable of recognizing foreignnucleic acids as non-self and inactivating or removing them from thecell. First recognized in plants and fungi, homology-dependent genesilencing mechanisms have now been shown to operate in a wide range ofeukaryotic organisms. In plants, this type of gene silencing may occurat the level of DNA, by inhibition of transcription, or at the level ofRNA, by enhanced RNA turnover. Both viral RNA and transgenes are subjectto these host surveillance systems, which are only poorly understood atthe molecular level. At the core of both silencing events resides amolecular mechanism that is able to recognize nucleic acid sequencehomology.

[0004] Transgene-induced gene silencing in plants was originallydescribed as the coordinated suppression of transgenes that sharesequence similarity (Depicker and Van Montagu, 1997). This phenomenon ismost often induced when multiple copies of a transgene are present at asingle locus. Silencing not only affects all genes in that locus, i.e.in cis, but also acts in trans, and additionally down-regulates theexpression of other, unlinked transgene(s). Silencing can also affectthe expression of endogenous genes, provided they have sequencesimilarity to the silencing transgene, a phenomenon referred to ascosuppression (Napoli et al., 1990; Van der Krol et al., 1990).

[0005] In plants, cases of transgene-induced gene silencing belong totwo different mechanistic classes: those that occur at the level oftranscription and those that are due to enhanced RNA turnover.Transcriptional gene silencing (TGS) requires sequence identity in thepromoter region and is associated with methylation and inactivation ofthe promoter sequences of the affected genes (Kumpatla et al., 1998). Inposttranscriptional gene silencing (PTGS), the (trans)genes remainactively transcribed but the steady-state RNA levels are highly reduceddue to sequence-specific RNA degradation. Some instances of PTGS areassociated with DNA methylation located in the transcribed portion ofthe genes (Ingelbrecht et al., 1994; 1999).

[0006] The expression level, number and configuration of the integratedtransgenes as well as developmental and environmental factors can allinfluence the occurrence of transgene-induced gene silencing.Importantly, transgene-induced gene silencing in plants is reversible,and in the absence of the silencer locus, expression of endogenous genesor other transgenes can be restored to normal. The changes in geneexpression are therefore not due to irreversible changes in DNA butrather are epigenetic.

[0007] PTGS behaves as a non-clonal event and, in agreement with this,it has been shown that a sequence-specific signal is involved in thesystemic spread of PTGS (Palauqui et al., 1997; Voinnet and Baulcombe,1997). These experiments allow differentiation of separate initiationand maintenance phases in PTGS and further suggest that a molecularsystem amplifies the silencing signal during the course of long-distancemovement of PTGS. Mutants that enhance (Dehio and Schell, 1994) orsuppress (Elmayan et al., 1998; Mourrain et al., 2000; Dalmay et al,2000) PTGS have been isolated in Arabidopsis thaliana but only two ofthe corresponding genes have been cloned (Mourrain et al., 2000; Dalmayet al, 2000). One of these has no significant similarity with any knownor putative protein (Mourrain et al., 2000) and the other is similar toa RNA-dependent RNA polymerase (RdRp; Mourrain et al., 2000; Dalmay etal, 2000). Establishment of PTGS in plants requires separatelyidentifiable initiation, spread, and maintenance phases, but theproteins involved in these pathways have not been characterized.

[0008] Plant virus studies have greatly contributed to the currentunderstanding of gene silencing in general and PTGS in particular.Applying the concept of pathogen-derived resistance, viral genes wereintroduced into plants and resulted in virus resistant phenotypes. Manyresistance phenotypes do not require the expression of a functionalprotein but are mediated at the level of RNA. It is now an establishedfact that a mechanism similar to PTGS is the underlying molecularmechanism in most of these cases (van den Boogaart et al., 1998).

[0009] Posttranscriptional silencing of an endogenous plant gene ortransgene can be triggered by replication of a recombinant virus thatcarries sequences homologous to these genes (Kumagai et al., 1995; Ruizet al., 1998). This process involves sequence-specific RNA turnover,similar to PTGS induced by transgenes, hence the term virus-induced genesilencing. Moreover, natural virus infection of non-transgenic plantscan induce a resistance mechanism that is strain-specific and targetedagainst RNA, similar to RNA-mediated resistance induced by (silenced)transgenes (Ratcliff et al., 1997; Covey et al., 1997). Transgene- andvirus-induced gene silencing are collectively described ashomology-dependent gene silencing because these mechanisms all targethomologous nucleic acid sequences. It was proposed thathomology-dependent gene silencing acts as a natural plant defensemechanism against invading DNA or RNA elements (Matzke and Matzke,1998).

[0010] The demonstration that plant viral proteins can suppress PTGSprovides direct evidence that PTGS functions as a host defense responsein plants (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschauand Carrington, 1998). At least 5 different proteins encoded byunrelated DNA and RNA viruses of plants have now been shown to act assuppressors of PTGS in Nicotiana benthamiana. Importantly, thesuppression phenotypes induced by these viral proteins are distinctindicating that separate steps of the host PTGS defense system aretargeted. For example, the potyviral helper-component proteinase(HC-Pro) can reverse the effects of PTGS in tissues that were previouslysilenced, whereas the 2b protein of Cucumber mosaic virus only affectsinitiation of PTGS (Voinnet et al., 1999). Although potyviral HC-Pro byitself is sufficient to suppress transgene-induced silencing, it appearsthat the potyviral P1 protein can enhance its ability to suppressvirus-induced gene silencing (Anandalakshmi et al., 1998; V. Vance). Thediscovery of viral suppressors of silencing phenomena is unique toplants. So far, no animal or fungal viruses have been shown to suppressPTGS in these organisms.

[0011] It has been proposed that ‘aberrant’ RNA molecules trigger PTGSin plants (Lindbo et al., 1993). The exact nature of this aberrant RNAis unknown but it could be double-stranded RNA (dsRNA) (Waterhouse etal., 1998), prematurely terminated transcripts, levels of RNA thatexceed a certain threshold, or some other unusual characteristic. TheseRNA molecules would serve as templates for an RNA-dependent RNApolymerase (RdRp) and lead to the production of short complementary RNAs(cRNA). These cRNAs would then anneal with homologous mRNAs or viralRNAs and the resulting double-stranded RNA would be degraded by doublestrand-specific RNases. This model accounts for the sequence-specificRNA turnover and several aspects of it are supported by experimentaldata. For example, an RdRp that is induced during viral infection hasbeen cloned in tomato (Schiebel et al., 1998) and small cRNAs haverecently been identified in transgenic plants that display PTGS(Hamilton and Baulcombe, 1999). The identification of a doublestrand-specific RNase in Caenorhabditis elegans and a RdRp-like proteinin Neurospora crassa, and recently in Arabadopsis, as essentialcomponents of PTGS-like mechanisms in these organisms (see below)provides further support for this hypothesis.

[0012] RNA-mediated genetic interference (RNAi) in C. elegans is aprocess that closely resembles PTGS in plants: both act at theposttranscriptional level and result in sequence-specific RNA turnover(Tabara et al., 1998; Montgomery and Fire, 1998). The trigger for RNAiin C. elegans is well characterized and consists of dsRNA (Sharp, 1999).RNA-specific silencing can be induced by locally injecting homologousdsRNA molecules in a few cells. Silencing then spreads from the site ofinjection into neighboring cells and tissues and is even transmitted tothe F1 progeny. The ability of silencing to move both in space and overtime strongly suggests that amplification of the silencing signal istaking place, similar to PTGS in plants.

[0013] Recently, several genes have been identified in C. elegans thatare required for this interference process. The MUT-7 gene encodes ahomolog of RNaseD, which is a double strand-specific RNase (Ketting etal., 19,99). The RDE-1 gene belongs to a family of genes that areconserved from plants to vertebrates and several members of this familyare required for gene silencing mechanisms in animal systems (Tabara etal., 1999). Interestingly, mutations in both these genes reactivatemobilization of endogenous transposons, suggesting that one function ofRNAi is transposon silencing. Sequence-specific inhibition of genefunction by dsRNA has also been demonstrated in trypanosomes, Drosophilaand planaria and has been used in these organisms as a method todetermine gene functions (Kennerdell and Carthew, 1998; Misquitta andPatterson, 1999; Sanchez Alvarado and Newmark, 1999).

[0014] Transgene-induced PTGS is termed ‘quelling’ in the fungus N.crassa (Cogoni and Macino, 1997a). Quelling-defective (qde) mutants ofN. crassa, in which transgene-induced gene silencing is impaired, havebeen isolated and could be classified in three qde complementationgroups (Cogoni and Macino, 1997b). Two QDE genes that belong to twodifferent complementation groups, have recently been cloned. The QDE-1gene encodes a protein that contains an RdRp-motif (Cogoni and Macino,1999a) and QDE-3 belongs to the RecQ DNA helicase family (Cogoni andMacino, 1999b).

[0015] As summarized above, there has been substantial progress in thegeneral understanding of PTGS in plants and its importance as part of ageneral defense system is now fully appreciated. However, all of thebiochemical pathways of PTGS and the enzymes that are involved have notyet been elucidated in plants. Insight into these mechanisms may comefrom analyzing mutants that are defective in PTGS. This approach hasalready been used with success in Neurospora and C. elegans and iscurrently also being followed for Arabidopsis. While this strategy isrelatively straightforward and will surely result in the identificationof genes that play a central role in this process, there are alsolimitations. For example, gene redundancies and possibly lethal,loss-of-function phenotypes might prevent identification of certaingenes. There are also practical problems in generating and screening asufficiently large number of mutants which limit this approach to modelplants such as Arabidopsis.

[0016] An alternative or complementary approach involves directlyidentifying the host factors that mediate PTGS. The identification ofviral proteins as suppressors of PTGS provides the necessary tools topursue this strategy.

[0017] Identification and characterization of proteins that interactwith a viral suppressor of PTGS will have an impact on understandingfundamentals of virus-host plant interactions, particularly on themechanisms that plants employ to combat viral infection and on thecounterdefensive strategies that viruses use to suppress or evade theseresponses. To date, viral suppression of PTGS is a process unique toplants. However, because PTGS is a defense mechanism that is conservedamong various eukaryotic kingdoms, the identified protein interactionsmight also shed light on the molecular mechanisms of silencing phenomenain other organisms.

[0018] In addition to significance for basic (plant) molecular virology,establishing the biochemical pathways of host defense responses willfacilitate the development of improved virus control strategies inplants. PTGS-based approaches for virus control are already in use butthe lack of a solid understanding of the phenomenon necessitates a moreempirical approach and has an uncertain outcome. Also, such approachesare currently limited because of their narrow range. Possible andrealistic improvements involve enhanced and more predictable triggeringand broadening the scope of the PTGS defense system.

[0019] Use of the method of the present invention will also contributeto plant genetic engineering in general. It is now clear that transgenesin plants (and other organisms) can be perceived as intrusive elementsand consequently are inactivated. Developing procedures that allowstable and predictable transgene expression is one of the challenges ofgenetic engineering. The monocot crop plants provide the most importantsource of food worldwide and offer great potential for improvementthrough genetic transformation, not only for traits related to foodproduction but also as recombinant expression systems for high valueproducts.

[0020] Finally, gene silencing can be used as a way to produce‘knock-out’ phenotypes in reverse genetic studies. This has already beensuccessfully applied in animal systems and its potential has beendemonstrated in plants. With an increasing number of genes beingdiscovered in sugarcane, many of which have no known function, it can beexpected that these approaches will become even more important in thefuture.

[0021] Thus, the yeast two-hybrid method of the present invention hasbeen used to unravel the pathway(s)of PTGS and plant defense responsesand novel, key proteins involved in this process have been identified.In doing so, a cDNA library from silenced plant tissues rather thannon-silence plant tissues has been used. These proteins and genes can beapplied towards regulating PTGS of transgenes, endogenous plant genes,and viral genes. Specific applications of the present invention includebut are not limited to, improved strategies for engineered virusresistance, increased expression of transgenes by inhibiting silencing,and modulation of silencing of native genes to obtain desirable traitsor in functional genomic studies.

SUMMARY OF THE INVENTION

[0022] The present invention includes a method of isolating nucleic acidencoding a plant polypeptide active in PTGS. As used throughout theapplication, plant may mean a mature plant, an embryonic callus, orother stages of plant development. It may also mean a portion of aplant, a plant tissue, or a plant cell.

[0023] The first step of a method of the above method involves selectinga bait nucleic acid which encodes a bait protein active in PTGS inplants or suppressive of PTGS in plants. After bait selection, a cDNAprey library may be prepared from a plant that actively exhibits PTGS atthe time of library generation. If an entire plant exhibiting PTGS isnot available, tissues in which PTGS is exhibited may be selected.

[0024] After the bait and prey are selected, a yeast two-hybrid assaymay be conducted with the bait and prey nucleic acids. Prey cDNA thatyields a true positive yeast two-hybrid assay result encodes apolypeptide active in PTGS in the plant. True positive status may beverified using methods known in the art, such as null controls, reversalof bait and prey, and in vitro and in planta studies of interactions.Such in vitro assay may include farwestern blot assays and pull downassays. They may be performed under plant physiological conditions toeliminate false negatives and false positives. In planta studies may beperformed in an embryonic callus or other plant tissue.

[0025] In an exemplary embodiment of the above method of the presentinvention, the bait nucleic acid comprises a sequence selected from SEQ.ID. NO. 1, SEQ. ID. NO. 3, SEQ. ID. NO. 5, SEQ. ID. NO. 7, SEQ. ID. NO.9, or SEQ. ID. NO. 11. The entire nucleic acids of these sequences maybe used or only portions thereof. Additionally, substituted nucleicacids and nucleic acids with similar identities may also be used.

[0026] In another exemplary embodiment the plant is a monocot,particularly sugarcane and more particularly Saccharum hybrid cultivarCP72-1210.

[0027] The present invention also includes several SrMV and sugarcanenovel proteins and nucleic acids. Novel nucleic acids are provided inSEQ. ID. NOS. 1,3,5,7,9 and 11. Novel amino acid sequences are providedin SEQ. ID. NOS. 2,4,6,8,10 and 12. It will be apparent to one skilledin the art that portions of these nucleic acids and proteins orpolypeptides may be used in various applications. Additionally, it willbe apparent to one skilled in the art that nucleic acids and proteins orpolypeptides with high similarity, particularly in regions related tofunctional domains, may also be substituted. The present inventionincludes such variations up to the point of disclosures already in theprior art. Each of these proteins is involved in PTGS either as anactivator or suppressor. Some proteins may fail to function alone andmay rather require or assist another protein for their PTGS-relatedfunctions.

[0028] The present invention additionally includes transgenic plantsincluding any of the above novel nucleic acids, proteins orpolypeptides. In particular, the invention includes a transgenic plantin which PTGS is suppressed that includes the nucleic acid of SEQ. ID.NO. 1 or the protein of SEQ. ID. NO. 2. It also includes a transgenicplant in which PTGS is enhanced that includes the nucleic acid of SEQ.ID. NO. 3 or the protein of SEQ. ID. NO. 4.

[0029] The invention additionally includes a method of increasing viralresistance in a plant in which a protein suppressive of PTGS in theplant is selected and the plant is transformed with a nucleic acidencoding the PTGS suppressive protein.

[0030] Another method of the invention involves a method of increasingexpression of a transgene in a plant by selecting a protein active inPTGS in the plant and transforming the plant with a nucleic acidencoding the protein active in PTGS.

[0031] Yet another method of the present invention involves suppressingexpression of a native gene in a plant by preparing a vector including anucleic acid with a sequence of the coding portion of the gene whereinthe nucleic acid, upon transcription, products an mRNA molecule doublestranded in the region corresponding the to the coding portion of thegene. A plant is then transformed with the vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] For a more complete understanding of the present invention andthe advantages thereof, reference is now made to the followingdescription taken in conjunction with the accompanying drawings, whereinlike reference numbers represent like parts, and which:

[0033]FIG. 1A provides the cDNA (SEQ. ID. NO. 1) and FIG. 1B providesthe amino acid (SEQ. ID. NO. 2) sequences for Sorghum Mosaic Virus(SrMV) P1/HC-Pro according to an embodiment of the present invention;

[0034]FIG. 2 is a Northern analysis of SrMV (coat protein) CP mRNA inwhich the lane labeled “S” includes mRNA derived from a plantposttranscriptionally silenced for SrMV CP; lanes labeled “RetransformedS” include mRNA from samples of the silenced plant which wereretransformed with either nptII alone or with nptII and SrMV P1/HC-Pro;the lane including mRNA from a plant that is not silenced is labeled“NS”; and the empty lane is labeled “b”;

[0035]FIG. 3 is a β-galactosidase filter lift assay on L40 yeasttransformed with various DNA binding and activation domain constructsand grown on media that does not select for interactions (−leu+zeo) ormedia that does select for transformation and interaction(−leu,−his+zeo); where the region labeled “A” represents yeasttransformed with SrMV HC-Pro bait+SrMV HC-Pro prey; the region labeled“B” represents yeast transformed with SrMV HC-Pro bait+empty prey; theregion labeled “C” represents yeast transformed with lamin bait+SrMVHC-Pro prey; and the region labeled “D” represents yeast transformedwith empty bait+SrMV HC-Pro prey; although the figure is presented inblack and white, growth in the regions marked A on both plants appearsblue while, on the −leu+zeo plate, growth in regions B-D appears red;

[0036]FIG. 4 is a gel analysis of the cDNA library cloned in pIVING1154;where Lane 1 contains a λPstI size marker; Lane 2 contains a lower bandof 0.8 kb which is the CP coding region amplified from the plasmid cDNAlibrary and a top band of 6.6-kb which is the plasmid vector; and Lane 3contains a SfiI digest of plasmid library showing the 6.6-kb vector andinserts visible as a smear with size range between 0.5 and 2.0 kb;

[0037]FIG. 5 is a β-galactosidase filter lift assay on L40 yeasttransformed with various DNA binding and activation domain constructsand grown on media that does not select for interactions (−leu+zeo) ormedia that does select for transformation and interaction(−leu,−his+zeo); where the region labeled “A” represents yeasttransformed with SrMV HC-Pro bait+SrMV HC-Pro prey; the region labeled“B” represents yeast transformed with empty bait+RNase H-like proteinprey; the region labeled “C” represents yeast transformed with laminbait +RNase H-like protein prey; and the region labeled “D” representsyeast transformed with SrMV HC-Pro bait+RNase H-like protein prey;although the figure is not presented in color, the regions marked A andD on both plates appear blue, while the regions marked B and C on the−leu+zeo plate appear red;

[0038]FIG. 6A is the cDNA sequence of the RNase H-like protein (SEQ. ID.NO. 3) and FIG. 6B is the encoded amino acid sequence (SEQ. ID. NO. 4),according to an embodiment of the present invention;

[0039]FIG. 7A is the cDNA sequence of the sugarcane RING zinc fingerprotein that interacts with SrMV HC-Pro (SEQ. ID. NO. 5); FIG. 7B is theencoded amino acid sequence (SEQ. ID. NO. 6) according to an embodimentof the present invention;

[0040]FIG. 8A is the cDNA sequence of the LRR (leucine-rich repeat)transmembrane protein kinase that interacts with SrMV HC-Pro (SEQ. ID.NO. 7); FIG. 8B is the encoded amino acid sequence (SEQ. ID. NO. 8)according to an embodiment of the present invention;

[0041]FIG. 9A is the cDNA sequence of a nucleic acid encoding thesugarcane 14-3-3 protein that interacts with the RNase H-like protein(SEQ. ID. NO. 9); FIG. 9B is the encoded amino acid (SEQ. ID. NO. 10),according to an embodiment of the invention;

[0042]FIG. 10A is the cDNA sequence of the sugarcane RING zinc fingerprotein that interacts with RNase H-like protein (SEQ. ID. NO. 11) andFIG. 10B provides the encoded amino acid sequence (SEQ. ID. NO. 12)according to an embodiment of the present invention;

[0043]FIG. 11A shows a 12% PA, CB stained gel; FIG. 11B shows a LargeS-AP probed blot; in parts of FIG. 10, the molecular weight lane isindicated as “MW”, lane 1 contains the E. coli expression product ofBugbuster™ (Novagen, Madison, Wis., affiliate of Merck KgaA, Darmstadt,Germany) Insoluble pET30 with no insert; lane 2 contains the E. coliexpression product of Bugbuster™ Insoluble pET30 with an SrMV HC-Proinsert; lane 3 contains one preparation of Ni column purified E. coliexpression product of Bugbuster™ Insoluble pET30 with an SrMV HC-Proinsert; and lane 4 contains a second preparation of Ni column purifiedE. coli expression product of Bugbuster™ Insoluble pET30 with an SrMVHC-Pro insert;

[0044]FIG. 12 shows a two hour exposure of a 15% SDS PAGE gel containingRNase H-like protein (lane labeled “RNase H”) labeled with 35S Cysteineaccording to the present invention; control lanes are provided in whichno DNA was used in the preparation procedure (lane labeled “No DNA”),andin which Luciferase DNA was used (lane labeled “Luciferase”); molecularweight markers are indicated;

[0045]FIG. 13 shows a 32 hour exposure of a farwestern blot probed underin vitro plant cell physiological conditions with a 35S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/HC-Pro” contains Ni column purified, His-S tagged SrMVHC-Pro protein produced in E. coli; the lane labeled “His-S” containsthe HIS-S tag only; the lane labeled “BSA” contains untagged bovineserum albumen; the lane labeled “HEWL” contains untagged hen egg whitelysozyme and the lane labeled “RNase A” contains untagged RNase A;

[0046]FIG. 14 shows a 6 hour exposure of a farwestern blot probed underin vitro plant cell physiological conditions with a ³⁵S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/HC-Pro” contains Ni column purified, His-S tagged SrMVHC-Pro protein produced in E. coli; the lane labeled “His-S/RNase H”contains Ni column purified, His-S tagged RNase H-like protein producedin E. coli; the lane labeled “His-S” contains the product in E. coli ofthe His-S tag only; the unlabeled lanes contain plant extracts which arefrom left to right, from: healthy sugarcane plant, SrMV infectedsugarcane plant, sugarcane plant transgenic for SrMV P1/HC-Pro,sugarcane plant transgenic for delta N 12 SrMV HC-Pro.; and the lanelabeled MWM contains molecular weight markers, which were used toproduce the size indicators on the side of the blot;

[0047]FIG. 15 shows a 32 hour exposure of a 15% SDS PAGE gel on which Nicolumn pull down products of a TNT 35S labeled protein/His-S taggedprotein physiological incubation were run; the top lane marker indicatesthe source of DNA used in a TNT procedure in to obtain 35S labeledprotein, where “RNase” indicates the use of RNase H-like protein cDNA,“No DNA” indicates a control in which no DNA template was provided, and“Luciferase” indicates a control in which Luciferase DNA was provided;the bottom lane marker indicates the His-S tagged product produced in E.coli, where “His-S/HC-Pro” or “HS/HC-Pro” indicates Ni column purifiedHis-S tagged SrMV HC-Pro protein, “His-S” indicates the His-S tag only,and “BSA” indicates untagged bovine serum albumen;

[0048]FIG. 16 shows a 3 hour exposure of a farwestern blot probed underin vitro plant cell physiological conditions with a 35S labeled RNaseH-like protein transcription and translation (TNT) product; the lanelabeled “His-S/14-3-3” contains Ni column purified, His-S taggedsugarcane 14-3-3 protein produced in E. coli; the lane labeled “His-S”contains the His-S tag only; the lane labeled “BSA” contains untaggedbovine serum albumen; the lane labeled “HEWL” contains untagged hen eggwhite lysozyme; and the lane labeled “RNase A” contains untagged RNaseA.

[0049]FIG. 17A depicts a DNA construct containing sense and antisensesequences in opposite directions and separated by an intron which,following transcription to mRNA, form a double strand due tosense/antisense sequence complementation shown in FIG. 17B;

[0050]FIG. 18 depicts the pMCG161 plasmid expression vector, accordingto one embodiment of the present invention;

[0051]FIG. 19 depicts the results of transient expression of constructsinserted in the pMGC161 plasmid in embryonic sugarcane calli where thecallus 1 is transformed with GUS under control of a Ubi promoter; callus2 is transformed with GUS under control of a Ubi promoter and DNA thatproduces double stranded GUS mRNA under control of a 35Sint promoter;callus 3 is transformed with the same constructs as callus 2 andadditionally with DNA that produces double stranded RNase H-like proteinmRNA under control of the 35Sint promoter; callus 4 is transformed withthe same constructs as callus 2 and additionally with SrMV P1/HC-Prounder the control of the Ubi promoter; callus 5 is transformed with thesame constructs as callus 2 and additionally with SrMV HC-Pro-delta N 12under control of the Ubi promoter for ease of plasmid construction toinclude a start ATG, the first N-terminal amino acids of the full lengthSrMV HC-Pro are deleted via deletion of 36 cDNA base pairs); and callus6 is transformed with the same constructs as callus 2 and additionallywith DNA that produces double stranded GFP under control of the 35Sintpromoter; although the figure is not provided in color, all darkerregions in the calli of the figure are blue while ligher regions arepinkish tan, as will be apparent to one skilled in the art;

[0052]FIG. 20 is a graphical representation of three experiments such asthat of FIG. 19; vertical bars represent the average of these threeindependent experiments while vertical line represent standard errors;treatment 1 represents transformation with GUS under control of a Ubipromoter; treatment 2 represents transformation with GUS under controlof a Ubi promoter and DNA that produces double stranded GUS mRNA undercontrol of a 35Sint promoter; treatment 3 represents transformation withthe same constructs as treatment 2 and additionally with DNA thatproduces double stranded RNase H-like protein mRNA under control of the35Sint promoter; treatment 4 represents transformation with the sameconstructs as treatment 2 and additionally with RNase H-like proteinunder control of the 35Sint promoter; treatment 5 representstransformation with the same constructs as treatment 2 and additionallywith SrMV P1 and HC-Pro under the control of the Ubi promoter; treatment6 represents transformation with the same constructs as callus 2 andadditionally with SrMV HC-Pro-delta N 12 under control of the Ubipromoter; and treatment 7 represents transformation with the sameconstructs as callus 2 and additionally with DNA that produces doublestranded GFP mRNA under control of the 35Sint promoter;

[0053]FIG. 21 depicts 4 embryonic calli transiently transformed withconstructs including, in callus 1, GUS under control of the 35Sintpromoter; in callus 2, GUS under control of the 35Sint promoter and DNAthat produces double stranded GUS mRNA also under control of the 35Sintpromoter; in callus 3, the same constructs as callus 2 and additionallyRNase H-like protein under control of the 35Sint promoter; in callus 4,the same constructs as callus 2 and additionally DNA that produces RNaseH-like protein mRNA under control of the 35Sint promoter; although thefigures is not presented in color, the darker colored calli, calli 1 and4 are blue while the lighter colored calli, calli 2 and 3 are pinkishtan was will apparent to one skiled in the art.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS OF THE INVENTION

[0054] The present invention includes a novel method for determinationof plant proteins active in PTGS or suppressive of PTGS. The inventionmay be used in both monocot and dicot plants, including sugarcane.

[0055] The methods of the present invention may be used, inter alia: (i)to identify, isolate and characterize cellular proteins that interactwith these PTGS suppressive viral proteins or plant proteins involved inPTGS using a yeast two-hybrid system; and (ii) to evaluate theseplant/viral protein interactions in vitro under physiological conditionsand in vivo in either transient studies or in transgenic plants, such assugarcane, expressing these viral proteins. Because many viral proteinssuch as P1 and HC-Pro are multifunctional proteins involved in variousaspects of plant/potyvirus interactions, the methods may also be used toassess the role of interacting host proteins in gene silencing. Themethods may be carried out using a combination of molecular genetics,immunological studies, transient antisense suppression studies, andplant transformation.

[0056] The overall method includes using a yeast two-hybrid system tosearch for plant proteins that interact with viral suppressors of PTGSto identify proteins involved in PTGS. These proteins or proteinsidentified as having a role in PTGS through other methods may then serveas bait to locate other proteins involved in PTGS. Because proteins thatinteract with other proteins involved in PTGS may be either suppressiveof or active in PTGS, the yeast two-hybird assays may identify bothtypes of proteins. Whether the proteins involved in PTGS are active inor suppressive of PTGS may be determined through a variety of methods,including identification of motifs with particular functions, comparisonwith known proteins, and in planta studies.

[0057] In the present invention the bait protein may be either active inPTGS or may function as a suppressor. The prey used is derived from anexpression library of the plant of interest. In an exemplary embodimentof the invention, the prey library is derived from a plant in which PTGSis occurring. This facilitates identification of proteins involved inPTGS because such proteins are actively being produced in the silencedplant and may therefore be better represented in the mRNA pool of asilenced plant than in a non-silenced plant.

[0058] After identification of an interacting prey, the bait and preyportions of the two-hybrid screen of the present invention may bereversed to help identify false positives. Comparison with controlsdesigned to look for activation of the reporter system absent either thebiat or prey may also be used to identify false positives.

[0059] Such yeast two-hybrid screens may then be followed by assays sucha farwestern blots or pull down assays to further determine whetheridentified proteins are false positives and also to characterize theirfunction. In planta physiological conditions may be used in such assays.

[0060] In planta studies may also be conducted using transiently orpermanently transformed embryonic calli transformed with either a testprotein or a DNA encoding a double stranded mRNA of the test protein(which induces PTGS of that protein) in order to further evaluate thesuppressive or effective role of the test protein in PTGS.

[0061] Proteins identified in one or more of the above screens may alsobe further characterized by identifying putative functional domains andalso be searching for overall cDNA and amino acid sequence similaritywith other proteins.

[0062] Often proteins and cDNAs identified using the above methodologiesmay be novel and patentable. In the present invention, five such novelproteins and cDNAs have been identified: the sugarcane 14-3-3 proteinand cDNA, the RNase H-like protein and cDNA, the sugarcane LRRtransmembrane protein kinase and cDNA, and the two sugarcane RING zincfinger proteins and cDNAs. Additionally, the SrMV P1/HC-Pro protein andcDNA used in some embodiments of the method of the present invention arealso novel.

[0063] The above cDNAs as well as other identified using themethodologies of the present invention may also be used to constructtransgenic plants, plant cells and plant tissues (collectively “plantentities”) in which PTGS is either enhanced or suppressed. Themethodologies used in the assay methods to generate embryonic calli andother methods know to the art may be used to construct these transgenicplant entities. Transformation may be transient or permanent, dependingupon the intended use of the transgenic plant entity.

[0064] Permanently transformed plant entities with increased PTGS may bevirus resistant. PTGS may be suppressed in other plants to allowincreased expression of a transgene for any of a variety of reasons,including improvement of plant health, adaptation to certain growingconditions, producing of a novel nutrient or vaccine, and production ofa protein later purified for medical or industrial uses. Finally, thePTGS-regulatory methods of the present invention may be used to inducePTGS of a particular gene, for instance by introducing a constructencoding dsRNA for a portion of the gene, thereby offering a novelmethod of producing knock-out plants.

[0065] The following examples are provided only to illustrate certainaspects of the invention and are not intended to embody the total scopeof the invention or any aspect thereof. Variations of the exemplaryembodiments of the invention below will be apparent to one skilled inthe art and are intended to be included within the scope of theinvention.

EXAMPLES Example 1 Nucleotide Sequence of Sorghum Mosaic Virus

[0066] SrMV is a member of the genus Potyvirus and can cause mosaicdisease and yield loss in poaceous plants such as sugarcane and sorghum(Shukla et al., 1994). A 2.0-kb region located at the 3′end of the SrMVstrain H genomic RNA which encompasses the 3′untranslated region, thecomplete open reading frame for the coat protein and part of the NIb ORFhas been previously sequenced(Yang and Mirkov, 1997). The remaining partof the SrMV genomic RNA has been sequenced in the present invention fromoverlapping RT-PCR products. From the combined sequences, a 9,581 bpconsensus was derived that contains a continuous ORF encoding a 3079amino acid putative polyprotein with ten gene products typical formembers of the genus Potyvirus (Ingelbrecht et al., in preparation). TheSrMV polyprotein consensus sequence was compared to that of Tobacco etchvirus (TEV-HAT), Maize dwarf mosaic virus (MDMV-Bu), Plum pox virus(PPV-D) and Pea seedborne mosaic virus (PSbMV-D) in a multiple sequencealignment using Clustal X. Based on this alignment, putative proteolyticcleavage sites were positioned in the SrMV polyprotein. (See FIG. 1.) Anovel nucleic acid (SEQ. ID. NO. 1) encoding a novel SrMV P1/HC-Proprotein (SEQ. ID. NO. 2) was developed.

Example 2 SrMV P1/HC-Pro Reverses PTGS

[0067] The potyviral HC-Pro is a multifunctional protein and harbors atleast 3 functional domains. The N-terminal part contains a highlyconserved KITC motif and is involved in aphid transmission (Thornbury etal., 1985; Atreya and Pirone, 1991). The central part is required forlong distance movement and virus amplification (Cronin et al., 1995;Kasschau et al., 1997) while the C-terminal part represents apapain-like proteinase involved in polyprotein processing (Carrington etal., 1990). In addition, potyviral HC-Pro is the determinant ofpotyviral synergistic interactions with other viruses (Pruss et al.,1997).

[0068] Potyviral HC-Pro can reverse the effects of PTGS in tissues thatwere previously silenced. This suppression phenotype has been observedfor the potyviral HC-Pro protein of three different potyviruses, TEV,PVY and PSbMV, suggesting that this particular function of potyviralHC-Pro may be conserved among different potyviruses (Voinnet et al.,1999). From this combined with the presence of conserved amino-acidsequence motifs in the SrMV HC-Pro, it was postulated that the SrMVHC-Pro might also act as a suppressor of PTGS in sugarcane. However, thesuppressor ability of SrMV HC-Pro could not be assumed based on sequencesimilarity alone and experiments were required to determine ifsuppressor activity was exhibited in sugarcane and the extent of suchsuppression.

[0069] Reversal of PTGS by SrMV P1/HC-Pro was demonstrated byretransforming a plant which is posttranscriptionally silenced for theCP transgene (Ingelbrecht et al., 1999, plant #16). The silenced plant#16 had originally been generated by selection on bialaphos. Embryogeniccallus generated from plant # 16 was therefore retransformed with eithernptII alone or with nptII and SrMV P1/HC-Pro, and transgenic plants wereselected on geneticin. As shown in the Northern blot analysis of the CPgene in FIG. 2, in 5 of the 6 plants retransformed with nptII and SrMVP1/HC-Pro, PTGS has been suppressed and the steady state levels of theCP mRNA are equivalent to the levels seen in a plant that is notsilenced (in FIG. 2, NS=plant #463 of Ingelbrecht et al., 1999). No suchreversal was seen in plants retransformed with nptII alone. Southern andNorthern analysis of nptII and SrMV P1/HC-Pro on these plants revealedthat the one plant retransformed with nptII and SrMV P1/HC-Pro in whichPTGS was not reversed was only transgenic for nptII (data not shown).

Example 3 The Yeast Two-Hybrid System

[0070] The yeast two-hybrid system has become a powerful tool for thestudy of protein-protein interactions in vivo. It can also be used tosearch an expression library for proteins that interact with a targetprotein. This latter application is being increasingly used with successin both animal (Kleiman and Manley, 1999) and plant systems (Kohalmi etal., 1998; Gindullis et al., 1999). Currently, high throughputtwo-hybrid procedures are being developed to catalogue protein-proteininteractions on a genome-wide scale (Walhout et al., 2000).

[0071] Before embarking on a screen, it is useful to study the behaviorof the target protein, termed bait, in the yeast cells. For example, oneshould demonstrate that the bait protein by itself does not activatetranscription of the marker genes that are used in the selection andscreening procedures. False positives can still be encountered followingthis precaution, but the great majority of these can be eliminated insubsequent steps. Finally, protein-protein interactions identified inthe yeast cells may be verified via independent experimental procedures,preferably in the original biological system or a physiologicallyequivalent environment.

[0072] A yeast two-hybrid screening procedure typically consists of thefollowing steps:

[0073] 1) Subclone the coding region of the target protein into a LexAor GAL4 binding domain vector to create a bait. In an example of thepresent method this bait is the SrMV HC-Pro sequence cloned intopHYBLexZeo to generate pIVING1281 or SrMV P1 cloned into pAS2 togenerate pIVING1088-6.

[0074] 2) Transform the bait into the yeast reporter strain to verifythat the expressed protein does not activate the reporter gene(s) byitself and is not toxic to the yeast cells. In an example of the presentmethod the yeast strain L40 is transformed with either pIVING1168,pIVING1281, or pIVING1088-6 alone, and also co-transformed with either apGAD424 derivative (pIVING1154) that lacks an insert, empty pHYBLexZeo,or pHYBLexZeo containing a non-specific bait (lamin). These sets oftransformed yeast strains function as negative controls for thescreening procedures as known in the art.

[0075] 3) Subclone a cDNA library of sequences into a GAL4 activatordomain vector to create a library of prey. In an example of the presentsystem, the cDNA library is derived from a transgenic sugarcane plantthat displays PTGS (Ingelbrecht et al., 1999, plant #16) and is clonedinto a modified pGAD424 derivative (pIVING1154), which constitutes theprey.

[0076] 4) Transform the yeast reporter strain carrying the bait plasmidwith the cDNA/prey library.

[0077] 5) Screen yeast bait and prey co-transformed colonies forexpression of a reporter gene that is fused to a GAL4 activatedpromoter. In an example of the present system, the yeast strain L40contains reporter constructs for expression of a β-galactosidase gene,as well as a reporter construct for the HIS gene. Cells in which baitand prey fusion proteins interact in the yeast nucleus will grow onminimal media in the absence of histidine and will produceβ-galactosidase enzyme activity at levels elevated from the negativecontrol cells.

[0078] 6) Verify positive interactions of co-transformants and eliminatefalse positives by reestablishing and retesting yeast strains, testingyeast strains which only contain the previously identified preyconstruct and testing the prey construct with empty bait or bait thatcontains a non-specific protein (such as lamin). If the prey constructactivates transcription of a reporter gene that is under regulation of aGAL4-inducible promoter under any of the above conditions, the prey maybe considered a false positive.

[0079] This method, as used in the examples of the present system, maybe used to isolate host proteins from sugarcane and other plants thatare involved in PTGS using the HC-Pro and P1 proteins from SrMV or otherproteins involved in PTGS as bait. Experiments using a library-scaleyeast two-hybrid screen with SrMV HC-Pro as bait have identified 441interacting proteins. At least 12 of these (including a protein withhigh similarity to a viral RNase H referred to herein as the “RNaseH-like protein”) have been confirmed to be true interactors. Additionalproteins involved in PTGS, such as a novel sugarcane 14-3-3 protein,have also been identified using the above method based on their abilityto interact with the RNase H-like protein used as bait.

Example 4 Self-Interaction with HC-Pro or P1 in a Yeast Two-HybridSystem

[0080] The yeast two-hybrid method of this invention may be used toidentify proteins from sugarcane that interact with HC-Pro or P1 ofSrMV. The two-hybrid procedure is based on the reconstruction of afunctional transcriptional activator such as GAL4 or LexA, whose DNAbinding domain (DBD) and transcription activation domain (AD) areexpressed on two different vectors (Fields and Song, 1989). In thepresent example, the bait protein, i.e. the SrMV HC-Pro, was fused tothe DNA-binding domain and the cDNA library was constructed in theactivation domain vector, which produces the prey. These vectors wereintroduced into the yeast strain L40, which has an endogenousβ-galactosidase (lacZ) reporter gene and the nutritional marker HIS3 forselection.

[0081] A 1.4-kb RT-PCR fragment encompassing the complete open readingframe of HC-Pro was amplified from SrMV virion particles and subclonedin pCR4-TOPO by T/A cloning (Invitrogen, Carlsbad, Calif.), yieldingpIVING1148-1. The complete HC-Pro ORF was cloned as a fusion proteininto the two-hybrid vectors pHYBLexZeo (bait) and pGAD424 (prey),yielding the plasmids pIVING1281 and pIVING1168, respectively. The P1ORF was subcloned as a 0.78-kb fragment in pAS2 yielding pIVING1088-6.The vector-insert junctions were confirmed by sequencing for allconstructs.

[0082] Recent studies have shown that potyviral HC-Pro proteins caninteract with themselves in a yeast two-hybrid system (Urcuqui-Inchimaet al., 1999; Guo et al., 1999), in agreement with an earlier proposalthat the potyviral HC-Pro is biologically active as a homodimer(Thornbury et al., 1985). The ability of the SrMV HC-Pro forself-interaction was tested in the present example. It was also verifiedthat the SrMV HC-Pro bait construct does not activate transcription ofthe lacZ gene, either by itself or in combination with empty bait orprey vectors. These latter experiments were also conducted for the P1bait construct pIVING1088-6. As shown in FIG. 3, the SrMV HC-Pro doesinteract with itself (A) in the two-hybrid system demonstrating thefunctionality of this construct. Secondly, no lacZ expression can beobserved in combination with either empty prey (B) or empty bait (D)vectors or a bait vector containing a non-specific protein (lamin; C).Therefore, SrMV HC-Pro does not activate transcription by itself and canbe used as bait to screen for interacting proteins. Similar results wereobtained for the SrMV P1 bait construct (data not shown).

Example 5 Development of a cDNA Library From Silenced Sugarcane

[0083] The development of SrMV-resistant sugarcane plants viatransformation with an untranslatable form of the capsid proteinsequence has been previously described and it has been demonstrated thatthe underlying resistance mechanism is related to PTGS (Ingelbrecht etal., 1999). As described in Ingelbrecht et al., 1999, plant #16 is arecovery plant that is immune to infection with SrMV after recovery fromthe initial infection and is posttranscriptionally silenced for the CPtransgene. Although the CP transgenes are actively transcribed, the CPsteady-state mRNA level is below the detection limit on an RNA gel blot(See Example 2 and FIG. 2).

[0084] Using poly(A)+RNA from silenced leaf tissue of this plant a cDNAfusion library was constructed in the prey vector pIVING 1154 using theSMART™ PCR cDNA library construction kit (offered by Clontech, Palo AltoCalif., a part of Beckton Dickinson, Franklin Lakes, N.J.). To createthe vector p1VING1154, pGAD424 was modified to allow directional cloningof the cDNA into asymmetric SfiI sites. The library has an estimated1.6×10⁶ primary clones. As shown in FIG. 4, the inserts range between0.5 to 2.0 kb with an average size of about 0.8 kb. Also, a 0.8-kbfragment containing the CP coding sequence could be readily amplifiedfrom the plasmid cDNA library indicating that low abundance mRNAs arerepresented.

Example 6 Library Scale Two-Hybrid Screen With HC-Pro Bait

[0085] The yeast strain L40 containing the SrMV HC-Pro bait constructwas transformed with the prey CDNA library representing the equivalentof approximately 1.6×10⁶ primary E. coli clones (see Example 4). About77 million primary yeast transformants were plated on selective media(−leucine,−histidine+zeocin) and 776 yeast colonies were recovered bythe fourth day of growth. Of these, 441 showed a range of light to heavyblue staining in the standard β-galactosidase colony-lift filter assay.To date, all of these double transformants have been colony purified,and all of the prey plasmids have been isolated. A RNase H-like protein,a RING zinc finger protein and LRR transmembrane protein kinase havebeen identified among the true positives. Some results of this screenand additional screens with SrMV PI or RNase H like-protein as the baitare depicted in Table 1. TABLE 1 Bait (DBD) Prey (AD) Interaction SrMVHC-Pro SrMV HC-Pro ++ SrMV P1 SrMV P1 −/+ SrMV HC-Pro SrMV P1 − SrMV P1SrMV HC-Pro − SrMV HC-Pro RNase H-like prot. +++ RNase H-like prot. SrMVHC-Pro +++ SrMV P1 RNase H-like prot. − RNase H-like prot. RNase H-likeprot. +++

Example 7 RNase H-Like Protein

[0086] One of the prey plasmids that was recovered during theexperiments of Example 5 was retransformed into the SrMV HC-Pro baityeast strain to reconfirm activation of the reporter genes. The plasmidencodes a protein with approximately 40% identity to a viral RNase H.This protein has been recovered several times using the methods of theseexamples. As shown in FIG. 5, the interaction with HC-Pro is very strong(D) and there is no interaction in combination with an empty baitplasmid or with a lamin bait plasmid (B and C, respectively).Furthermore, the RNase H-like gene is in the correct reading frame withrespect to the fusion protein, so this is a true interactor. The cDNAsequence (SEQ. ID. NO. 3) for a nucleic acid encoding the RNase H-likeprotein and its amino acid sequence (SEQ. ID. NO. 4) are provided inFIG. 6.

Example 8 RING Zinc Finger Protein That Interacts With HC-Pro

[0087] Another protein identified as a true positive in HC-Prointeraction screens is a RING zinc finger protein. A partial cDNAsequence of a nucleic acid encoding this protein is provided in FIG. 7A(SEQ. ID. NO. 5). The encoded amino acid sequence is included as FIG. 7B(SEQ. ID. NO. 6). The nucleic acid sequence is sufficient to identifynucleic acids encoding the protein, but likely lacks approximately 300bp at the 5′ end in the figure. However, because a protein encoded bythe possibly truncated nucleic acid sequence was identified in atwo-hybrid screen, a nucleic acid with the sequence provided must encodea sufficient portion of the protein to allow interaction with SrMVHC-Pro. With the information disclosed in this invention, it is possiblefor a person skilled in the art to isolate the full length cDNA andprotein sequence.

Example 9 LRR Transmembrane Protein Kinase

[0088] Another protein identified as a true positive in SrMV HC-Proyeast two-hybrid screens is an LRR transmembrane protein kinase. Apartial cDNA sequence of a nucleic acid encoding this protein isprovided in FIG. 8A (SEQ. ID. NO. 7). The encoded amino acid sequence isincluded in FIG. 8B (SEQ. ID. NO. 8). The sequence is sufficient toidentify nucleic acids encoding the protein, but likely lacksapproximately 1 kb at the 5′ end in the figure. However, because aprotein encoded by the possibly truncated nucleic acid sequence wasidentified in a two-hybrid screen, a nucleic acid with the sequenceprovided must encode a sufficient portion of the protein to allowinteraction with SrMV HC-Pro. With the information disclosed in thisinvention, it is possible for a person skilled in the art to isolate thefull length cDNA and protein sequence.

Example 10 Library Scale Two-Hybrid Screen With RNase H-Like Protein asBait

[0089] A library scale two-hybrid screen similar to that of Example 5was conduced using the same prey library (described in Example 4) withthe RNase H-like protein as bait. Using the RNase H-like protein asbait, at least two true positives for prey/host proteins were identifiedand further characterized. The results of some of these assays as wellas assays with SrMV HC-Pro and SrMV P1 as bait are depicted in Table 2.TABLE 2 Bait (DBD) Prey (AD) Interaction SrMV HC-Pro SrMV HC-Pro ++RNase H-like prot. SrMV HC-Pro +++ SrMV P1 RNase H-like prot. − RNaseH-like prot. RNase H-like prot. +++ RNase H-like prot. Sugarcane 14-3-3+++ SrMV HC-Pro Sugarcane 14-3-3 − SrMV P1 Sugarcane 14-3-3 −

Example 11 Sugarcane 14-3-3 Protein

[0090] One protein identified through its ability to interact with theRNase H-like protein is a sugarcane 14-3-3 protein. The sequence of anucleic acid encoding the sugarcane 14-3-3 protein is provided in FIG.9A (SEQ. ID. NO. 9). The encoded amino acid sequence is provided in FIG.9B (SEQ. ID. NO. 10). The role of another 14-3-3 protein in signaltransduction in the dicot Arabidopsis has recently been described inSehnke et al., 2002. A similar role in monocots is suggested by thedisclosed in this invention. Specifically, 14-3-3 proteins exhibit aphosphorylation-dependent association with proteins in dicots. The RNaseH-like protein of the present invention contains a serine with a 99.1%chance of being phosphorylated. This serine is also located in a goodconsensus 14-3-3 protein binding site. More specifically, the putativephosphorylation/binding site is VIQNpSPPDL (wherein “pS” designatesphosphoserine) beginning at amino acid 48 of the protein in FIG. 9B.Binding of sugarcane 14-3-3 and the RNase H-like protein in order toachieve a functional dimer (or as part of a functional complexcontaining other proteins) is also suggested by the absence of DNase orRNase activity of either protein in isolation. As shown in Table 2,14-3-3 does not interact noticeably with SrMV HC-Pro or SrMV P1.

Example 12 RING Zinc Finger Protein That Interacts With RNase H-LikeProtein

[0091] Another protein identified as a true positive in RNase H-likeprotein interaction screens is a RING zinc finger protein. This is notthe same RING zinc finger protein identified as interacting with SrMVHC-Pro. A partial cDNA sequence of a nucleic acid encoding this proteinis provided in FIG. 10A (SEQ. ID. NO. 11). The amino acid sequenceencoded by the nucleic acid is provided in FIG. 10B(SEQ. ID. NO. 12)

Example 13 DNA Sequence Analyses of the Prey Genes Identified in theTwo-Hybrid Screen

[0092] Prey sequences that have been verified by the above screeningprocedures may be DNA sequenced utilizing standard fluorescence-basedthermocycle sequencing methods, and restriction maps created. Thissequence information may be utilized to search the genetic databaseswith BLASTx and BLASTp to determine sequence similarity with known genesor proteins. In cases where similarity is clear, one may verify that thesequence under investigation is inserted in the appropriate readingframe in the prey vector. If the sequence is not in the appropriatereading frame, the prey can be considered a false positive.

Example 14 Characterization of the Prey Nucleic Acids Identified in theTwo-Hybrid Screen for the Ability of Their Corresponding Proteins toInteract With SrMV HC-Pro Protein or Other Bait Protein in vitro UnderPhysiological Conditions

[0093] Although prey and bait combinations identified in the two-hybridscreen of the present invention may represent proteins which interact inyeast cells, several caveats of the assay may produce results which arenot indicative of interactions that occur in planta. The yeasttwo-hybrid system assay requires that the bait and prey GAL4 fusionproteins both be imported into the yeast nucleus, which biochemically isa much more reducing environment than the yeast cytoplasm. This may leadto different patterns of protein folding than might otherwise occur forproteins whose operating environment is a more oxidative cytoplasm.Additionally, protein fragments placed in an unnatural position in ayeast two-hybird assay (e.g. an N term region in the C term of thefusion protein) may fail to fold or function properly because ofpositional effects.

[0094] Given that in vivo studies often require specialized reagents inthe present method, an initial in vitro screening procedure in whichpotential protein interactions are verified under physiologicalconditions may be used to save time and expense associated with planttransformations in the case of false positives. However, it does remainpossible that in vitro conditions may lack certain requirements forinteraction found only in living cells and thus generate falsenegatives. Therefore, in some instances it may be desirable to proceedwith in planta studies without or despite results in vitro. Such anapproach is within the scope of the present invention.

[0095] If in vitro studies are preformed, host/prey proteins identifiedin the two-hybrid screen may be further tested for their ability tointeract with SrMV P1 and SrMV HC-Pro or other proteins involved in PTGSin vitro under physiological conditions (i.e. pH 6.8 and 0.1 M ionicstrength) as either polyhistidine, S-tag, or glutathione-s-transferase(GST) fusion-proteins. A series of vectors from Novagen, Inc. (Madison,Wis., affiliate of Merck KgaA, Darmstadt, Germany) including pET15b,pET29a,b,c and pET30a,b,c, allow the construction of N-terminal orC-terminal polyhistidine fusion proteins, which in the case of pET29 andpET30 derivatives may also contain S-tags. Pharmacia Corp. (Peapack,N.J.) also produces pGEX2, pGEX3 and pGEX4 derivatives in which GSTfusion proteins may be produced. These vectors allow the specificpurification of “tagged” proteins that have either polyhistidine tags(using Ni+-chelated resin), S-tags (using S-agarose), or GST-tags (usingreduced glutathione-conjugated resins). Bait and prey inserts identifiedfrom the two-hybrid screen may be subcloned into one of these vectors.Other vectors encoding tag regions may also be used.

[0096] One approach is to construct two vector types, if possible, forthe bait as well as for each prey that has been identified and toproduce GST- and polyhistidine tagged proteins in E. coli. These taggedproteins may be purified with kits available from Pharmacia, Novagen, orother sources, or using methods known to the art and appropriate for theselected tag. His-S tagged SrMV HC-Pro produced in the pET30 vectorpurified with a Ni column is shown in FIG. 11 in lanes 3 and 4. As FIG.11 indicates, tagged protein is produced only when an insert is presentin the vector and may be largely purified using a Ni column.

[0097] Radiolabelled SrMV HC-Pro, SrMV P1, RNase H-like protein or otherbait proteins of interest may be produced by translation of theircorresponding transcripts in rabbit reticulocyte lysate. Using the TNSmethod, transcripts may be generated in a T7 in vitro transcriptionsystem or other system known to the art. Subsequent translation in thepresence of S-35-labeled methionine or cysteine allows radiolabeling ofthe bait protein. FIG. 12 shows that RNase H-like protein produced usingthe above methodology is, in fact, radiolabeled. A small amount of thistranslated lysate may then be incubated with the tagged prey proteinbound to its corresponding resin, and retention of radiolabeled baitprotein may be assayed with SDS-PAGE gels and autoradiograms. Positivecontrols can include tagged bait protein, and negative controls caninclude the tags themselves, as well as the resins without any otherproteins. These procedures or various modifications thereof readilydetermined by one skilled in the art allow one to determine whetherprospective prey proteins are capable of interacting with either one orboth viral proteins in vitro under physiological conditions.

Example 15 Farwestern Blots and Other Assays Indicate Interaction ofVarious Proteins

[0098]FIG. 13 shows a farwestern blot of His-S tagged SrMV HC-Pro probedwith 35S labeled RNase H-like protein. Both proteins were producedaccording to the methods of the above examples. A comparison of bindingof labeled RNase H-like protein to the His-S tagged SrMV HC-Pro to thevarious controls indicates that the interaction is specific between thetwo proteins and also that it occurs in conditions approximating plantphysiological conditions.

[0099]FIG. 14 shows a farwestern blot of His-S tagged SrMV HC-Pro andRNase H-like protein and a plant extracts probed with ³⁵S labeled RNaseH-like protein. A comparison of the lanes indicates that the RNase Hinteracts with itself and SrMV HCPro and with other sugarcaneproteins-from left to right-healthy sugarcane plant, SrMV infectedsugarcane plant, sugarcane plant transgenic for SrMV P1/HC-Pro,sugarcane plant transgenic for SrMV delta N 12 HC-Pro. An extra band inthe three plants expressing SrMV HC-Pro that is slightly smaller thanthe tagged SrMV HC-Pro may be seen in FIG. 14.

[0100]FIG. 15 shows the results of a Ni column pull down of His-S taggedprotein and its binding partner. The proteins attached to the Ni columnwere removed and run on an SDS PAGE gel. The results indicate that His-Stagged SrMV HC-Pro and not control proteins pulled down labeled RNaseH-like protein. Label controls were not pulled down. This indicates thatthe SrMV HC-Pro/RNase H-like protein interaction is specific and viableunder physiological conditions.

[0101]FIG. 16 shows a farwestern blot of His-S tagged sugarcane 14-3-3probed with 35S labeled RNase H-like protein. A comparison of binding oflabeled RNase H-like protein to the His-S tagged sugarcane 14-3-3 to thevarious controls indicates that the interaction is specific between thetwo proteins and also that it occurs in conditions approximatingphysiological conditions.

Example 16 HC-Pro Deletion Mutant and Interaction With RNase H-LikeProtein

[0102] In order to better determine the region of SrMV HC-Proresponsible for interaction with the RNase H-like protein, a series ofdeletion mutants were created and their interaction was tested in yeasttwo-hybrid and farwestern blot assays as described above. The results ofthese experiments are summarized in Table 3 and indicate that the Cterminal portion, including the last 10 kDa of the HC-Pro protein arerequired for interaction with the RNase H-like protein while the Nterminal region up to 7 kDa is not necessary. TABLE 3 HC-Pro segmentAssay Interaction Full length Yeast two-hybrid +++ ←----------------→Full length Farwestern +++ ←----------------→ N 1.3 kDa deletion Yeasttwo-hybrid +++   ←-------------→ N 7.0 kDa deletion Yeast two-hybrid +++   ←---------→ C 10 kDa deletion Farwestern − ←----------→ C 20 kDadeletion Farwestern − ←-----→

Example 17 Complete Cloning of Partial cDNA Prey Clones

[0103] Candidate clones whose corresponding protein sequences interactwith SrMV HC-Pro or other bait proteins in vitro under physiologicalconditions may be completely cloned with 5′ RACE procedures or otherprocedures known to the art.

Example 18 Production of Antiserum

[0104] To facilitate in vivo co-immunoprecipitation studies furtheroutlined below, antisera recognizing SrMV HC-Pro or another proteininvolved in PTGS may be produced. Using a pET or PGEX expression plasmidconstruct as described above or another 6×his construct known to theart, one may produce 6×his-tagged SrMV HC-Pro or other protein in E.coli, purify the fusion protein as described above, and utilize theprotein as an antigen with rabbits or other animals to producepolyclonal antisera. Similarly, SrMV P1 and host proteins may also beutilized to produce antisera, depending upon the potential utility ofthe resultant serum. Other antisera production techniques may also beused to produce polyclonal or, where useful, monoclonal antibodies.

Example 19 Transgenic Plant Studies

[0105] Because of its specific mode of action, the SrMV P1/HC-Pro andother proteins identified as involved in PTGS using the above two-hybridand in vitro methods may target one or more factors that are expressedand functional in silenced tissue. This may be confirmed in planta.Because the cDNA library in the above examples was constructed from aplant harboring a PTGS-silenced SrMV coat protein sequence, and theplant is resistant to SrMV, one cannot readily utilize mechanicaltransmission of SrMV as a source of SrMV HC-Pro or other viral proteinsin such plants, and thus may resort to transgenic methodologies.

[0106] Plant transformation studies demonstrate the feasibility of thisapproach. To date, SrMV HC-Pro transformants and SrMV P1/HC-Protransformants have been confirmed via Southern and Northern analyses.The plants were produced via particle gun transformation of embryogeniccallus, as previously described (Ingelbrecht et al., 1999). Threedifferent plant transformation vectors have been constructed: (1)pIVING1023 which contains the SrMV P1/HC-Pro polyprotein sequence, inwhich SrMV HC-Pro may be expected to be cleaved out by the SrMV P1protease in planta, (2) pIVING1002 which contains only the SrMV P1sequence, and (3) pIVING991-1 which contains only the SrMV HC-Prosequence. In the present example, two selectable markers for sugarcanetransformation have been used, the nptII gene in combination withgeneticin, and the bar gene for selection with bialaphos. Bialaphosselection may be used on bar transformed cells, and geneticin selectionmay be used on bar transgenic cells to select for second transformationevents of previously transformed tissue. Other transformation mechanismsand selection systems known to the art may also be used. Suchsequentially transformed plants may serve as a source of material for inplanta studies.

[0107] In a further example, the transgenic plant #16 that was used as asource for construction of the prey cDNA library above, was utilized togenerate embryogenic callus. Plant #16 was previously transformed withthe bar gene, and in this example was transformed again with the SrMVP1-HC-Pro construct using geneticin selection. Because SrMV P1 has beendescribed as an enhancer of the PTGS-inhibition by SrMV HC-Pro, thecapsid message levels may be higher in SrMV P1-HC-Pro transformed #16plants as compared to SrMV HC-Pro transformed #16 plants. In addition tohaving a system of modulated PTGS-suppression, these transgenic plantsalso allow verification in planta of the interactions between the viralproteins and the identified prey proteins, utilizing SrMV HC-Prospecific antiserum in standard co-immunoprecipitation methodologies.

[0108] Sequence homology of the isolated host proteins with knownproteins, if present, may be used to postulate their biological functionand whether or not they are involved in PTGS in a manner similar to thatemployed with the genes identified in mutagenesis studies of Neurospora,C. elegans and Arabadopsis.

[0109] One approach for determining functions of the isolated genes maybe based on creating suppression phenotypes for these genes in sugarcanevia genetic transformation. Efficient triggering of gene suppression canbe achieved by simultaneous expression of sense and antisense constructsor constructs designed to produce dsRNA as demonstrated in tobacco, riceand Arabadopsis (Waterhouse et al., 1998; Smith et al., 2000; Chuang andMeyerowitz, 2000). See also FIG. 17 for depictions of the basicstructure of DNA and resulting mRNA for such constructs. Such constructsmay be created for each of the isolated host genes and introduced inplants that display PTGS of a capsid protein transgene, such as plant#16. The transformation methodologies described above can be followed togenerate stably transformed plants. Alternatively, a protoplast-basedsystem can be used in combination with electroporation or PEG-mediatedtransformation. Expression of the capsid protein will be analyzed andreversal of silencing may indicate that the PTGS mechanism in thesetransgenics is impaired and that the transformed host genes are involvedin PTGS. Example 20

In Planta Studies of HC-Pro and RNase H-Like Protein

[0110] Embryonic sugarcane calli were bombarded with various constructsaccording to the above examples in order to provide transient expressionto verify the role of HN-Pro and RNase H-like protein in PTGS. Doublestranded mRNA relating to the various proteins was generated usingconstructs such as those show in FIG. 17 inserted into the vector ofFIG. 18.

[0111]FIG. 19 shows that GUS under a Ubi promoter alone is expressed inembryonic callus. Introduction of a double stranded GUS mRNA reduces itsexpression by activating PTGS of GUS mRNA. However expression isrestored by both introducing SrMV P1-HC-Pro into the callus and byinducing PTGS of the RNase H-like protein using a double stranded mRNAof that protein. Average results for three separate experiments of thisnature are depicted in FIG. 20 and show that statistically. significantresults are consistently obtained. Overall, these experiments confirmthat SrMV P1 and SrMV HC-Pro inhibits PTGS and that the RNase H-likeprotein is involved in effecting PTGS.

[0112]FIG. 21 illustrates the PTGS effect of the RNase H-like proteineven more clearly. In these figures calli transiently transformed withGUS exhibit the protein. This protein production is severely disruptedby subsequent or simultaneous transformation with DNA that producesdouble stranded GUS mRNA, which triggers PTGS. PTGS is not turned off bythe transformation of the GUS/double stranded GUS calli with RNase H.However, it is severely hampered by introduction of DNA that producesdouble stranded mRNA for RNase H-like protein, which induces PTGS ofRNase H-like protein itself. These experiments demonstrate that theRNase-H like protein plays a role in PTGS. These results are summarizedin Table 4. TABLE 4 Transformation Constructs Callus Color 35S-int/GUSBlue 35S-int/GUS + 35S-int/dsGUS Not Blue 35S-int/GUS + 35S-int/dsGUS +Not Blue 35S-int/RNase H-like protein 35S-int/GUS + 35S-int/dsGUS + Blue35S-int/ds RNase H-like protein

[0113] All references mentioned in any portion of the foregoingspecification are incorporated by reference herein.

1 12 1 2103 DNA Sorghum mosaic virus strain H 1 gaattcgccc ttatttcagccatggcagga gcatggaaca ctgtgactta caagtggagg 60 ccgaatttgg acaacgcaagagatgttcga aaagtgatgg aacattttgc agcaaagcac 120 caagtttatg atgcaaagcgtgcagcagag cataacagta gaatccttcg caggactttt 180 gtacaagaaa ttgcaaaagcacctgaagag aagacttcat acaaacctca ggtgtgggtc 240 gaaaaacaag ataacaatccaacgatccat ctacactatg ttagattcaa aaacaaggag 300 aaaaagatat tacccgagatcactccagga tcggtcgcaa agttaacgcg acggatactt 360 gagctcagca aaactacaaagcttgaagtt gaactcattg gtaagaaaag acgcaaatcg 420 actaaactag caatcaaaaggcgcagaaac agagagtatt tgcattgtga aactagacac 480 gaaacaaaca aatttaagcgcgttgacatc aacatagaac gacactggtt tccacttgtg 540 aagaagattt caaagtgctatagtcacata tcaccaagaa tgtacaaaaa catgagcaaa 600 ggcgacagtg ggttaacattcatccagaat ggtgagttat ttataatccg aggaaaacga 660 gatggcgtcc tacttaatagtatcaccaat gaaactcgaa ttaatgaaat aacttatttt 720 agcgatgctc aggcgaacgacttctggcga ggttacacag atcatatggt cgaaaatagg 780 ttaatttcta caactcatacagaacacata cccacaataa atttagagaa gtgtggaaag 840 aggatggcat tgttagagatattgtttcac tccacattca aaattacgtg taagcactgt 900 aacaatgacg atcttgaactatcggatgat gagtttggag aaagactata taagaactta 960 atcagaattg aagaaaagcaaaaagaatat ttagctgaag atcaaaagct taagcgaatg 1020 atatcctttc tgaaggatagatgcaatcca aaatttgagc atttaccatt attatggcag 1080 gtcgctgaaa caattggacattacactgat aatcaagcaa aacagatcct tgaagttaat 1140 gaagcgctca taaaagtgaacactctttct gttgaagatg cagtcaaagc tagcgcatcg 1200 ttgctagaga tttcaagatggtacaagaat aggaaagaat catcgaaaga aggtacactt 1260 agtacattca ggaataaaatttcacctaaa agtactatta atacagcact gatgtgtgat 1320 aatcagctcg atacaaatggtaacttccta tggggaaaga gagaatatca tgccaagcga 1380 ttctttacaa actattttgaagctgttgat ccaaaagaca cgtatgaaaa gcatgttact 1440 cggttcaatc caaatggtcaacgcaaactt tcgattggaa aactagttat cccattagac 1500 ttccagaaga ttcgtgaatcatttataggt gttcaagttc aaaaacaagc aattagtaga 1560 gcgtgcttaa gtaaaatcgaaaataattac atataccctt gctgttgtgt aactacagaa 1620 tttggtcaac cggtttattcagagatcatt ccaccaacta aaggtcatat tactattgga 1680 aattcgaccg acccaaaaattgtggatttg cctaattccg acccaccaat gatgtacata 1740 gcgaaagatg gttattgttatttgaatata tttttagctg ctctgataaa cgtcaatgaa 1800 gattcagcaa aagattacacaaagtttttg cgtgatgaac taattgaaag acttggaaag 1860 tggccaaaac tcaaagacgtggcgacagca tgttatgcat tatcagtaat gtttccagaa 1920 attaagaacg cggagcttccacaaatacta gtggaccacg aacataaaac catgcatgtg 1980 atagattcgt acggatctctcagtgttggc ttccacatac tcaaagcgaa cacaatagga 2040 caattaatca aaatgcaatatgaatccatg gaaagtgaaa tgagagagta tgtagtcggt 2100 tag 2103 2 694 PRTSorghum mosaic virus strain H 2 Met Ala Gly Ala Trp Asn Thr Val Thr TyrLys Trp Arg Pro Asn Leu 1 5 10 15 Asp Asn Ala Arg Asp Val Arg Lys ValMet Glu His Phe Ala Ala Lys 20 25 30 His Gln Val Tyr Asp Ala Lys Arg AlaAla Glu His Asn Ser Arg Ile 35 40 45 Leu Arg Arg Thr Phe Val Gln Glu IleAla Lys Ala Pro Glu Glu Lys 50 55 60 Thr Ser Tyr Lys Pro Gln Val Trp ValGlu Lys Gln Asp Asn Asn Pro 65 70 75 80 Thr Ile His Leu His Tyr Val ArgPhe Lys Asn Lys Glu Lys Lys Ile 85 90 95 Leu Pro Glu Ile Thr Pro Gly SerVal Ala Lys Leu Thr Arg Arg Ile 100 105 110 Leu Glu Leu Ser Lys Thr ThrLys Leu Glu Val Glu Leu Ile Gly Lys 115 120 125 Lys Arg Arg Lys Ser ThrLys Leu Ala Ile Lys Arg Arg Arg Asn Arg 130 135 140 Glu Tyr Leu His CysGlu Thr Arg His Glu Thr Asn Lys Phe Lys Arg 145 150 155 160 Val Asp IleAsn Ile Glu Arg His Trp Phe Pro Leu Val Lys Lys Ile 165 170 175 Ser LysCys Tyr Ser His Ile Ser Pro Arg Met Tyr Lys Asn Met Ser 180 185 190 LysGly Asp Ser Gly Leu Thr Phe Ile Gln Asn Gly Glu Leu Phe Ile 195 200 205Ile Arg Gly Lys Arg Asp Gly Val Leu Leu Asn Ser Ile Thr Asn Glu 210 215220 Thr Arg Ile Asn Glu Ile Thr Tyr Phe Ser Asp Ala Gln Ala Asn Asp 225230 235 240 Phe Trp Arg Gly Tyr Thr Asp His Met Val Glu Asn Arg Leu IleSer 245 250 255 Thr Thr His Thr Glu His Ile Pro Thr Ile Asn Leu Glu LysCys Gly 260 265 270 Lys Arg Met Ala Leu Leu Glu Ile Leu Phe His Ser ThrPhe Lys Ile 275 280 285 Thr Cys Lys His Cys Asn Asn Asp Asp Leu Glu LeuSer Asp Asp Glu 290 295 300 Phe Gly Glu Arg Leu Tyr Lys Asn Leu Ile ArgIle Glu Glu Lys Gln 305 310 315 320 Lys Glu Tyr Leu Ala Glu Asp Gln LysLeu Lys Arg Met Ile Ser Phe 325 330 335 Leu Lys Asp Arg Cys Asn Pro LysPhe Glu His Leu Pro Leu Leu Trp 340 345 350 Gln Val Ala Glu Thr Ile GlyHis Tyr Thr Asp Asn Gln Ala Lys Gln 355 360 365 Ile Leu Glu Val Asn GluAla Leu Ile Lys Val Asn Thr Leu Ser Val 370 375 380 Glu Asp Ala Val LysAla Ser Ala Ser Leu Leu Glu Ile Ser Arg Trp 385 390 395 400 Tyr Lys AsnArg Lys Glu Ser Ser Lys Glu Gly Thr Leu Ser Thr Phe 405 410 415 Arg AsnLys Ile Ser Pro Lys Ser Thr Ile Asn Thr Ala Leu Met Cys 420 425 430 AspAsn Gln Leu Asp Thr Asn Gly Asn Phe Leu Trp Gly Lys Arg Glu 435 440 445Tyr His Ala Lys Arg Phe Phe Thr Asn Tyr Phe Glu Ala Val Asp Pro 450 455460 Lys Asp Thr Tyr Glu Lys His Val Thr Arg Phe Asn Pro Asn Gly Gln 465470 475 480 Arg Lys Leu Ser Ile Gly Lys Leu Val Ile Pro Leu Asp Phe GlnLys 485 490 495 Ile Arg Glu Ser Phe Ile Gly Val Gln Val Gln Lys Gln AlaIle Ser 500 505 510 Arg Ala Cys Leu Ser Lys Ile Glu Asn Asn Tyr Ile TyrPro Cys Cys 515 520 525 Cys Val Thr Thr Glu Phe Gly Gln Pro Val Tyr SerGlu Ile Ile Pro 530 535 540 Pro Thr Lys Gly His Ile Thr Ile Gly Asn SerThr Asp Pro Lys Ile 545 550 555 560 Val Asp Leu Pro Asn Ser Asp Pro ProMet Met Tyr Ile Ala Lys Asp 565 570 575 Gly Tyr Cys Tyr Leu Asn Ile PheLeu Ala Ala Leu Ile Asn Val Asn 580 585 590 Glu Asp Ser Ala Lys Asp TyrThr Lys Phe Leu Arg Asp Glu Leu Ile 595 600 605 Glu Arg Leu Gly Lys TrpPro Lys Leu Lys Asp Val Ala Thr Ala Cys 610 615 620 Tyr Ala Leu Ser ValMet Phe Pro Glu Ile Lys Asn Ala Glu Leu Pro 625 630 635 640 Gln Ile LeuVal Asp His Glu His Lys Thr Met His Val Ile Asp Ser 645 650 655 Tyr GlySer Leu Ser Val Gly Phe His Ile Leu Lys Ala Asn Thr Ile 660 665 670 GlyGln Leu Ile Lys Met Gln Tyr Glu Ser Met Glu Ser Glu Met Arg 675 680 685Glu Tyr Val Val Gly Glx 690 3 937 DNA Saccharum hybrid cultivarCP72-1210 3 ggccattatg gccggggaga acactgtatg aagaacatgg ccgcttcttatagtcaagac 60 gcgcagctcg atctcgtact tcctgacgct cccgtcgatg ctagcgcgtctcgttctgaa 120 cattcatctc agcttgctag ctctaactgg agatctgtga ttcaaaactccccacctgat 180 ctcctatgcg gatgcggtag accggcaatt aggcgcacgg cagagactgcgaagaacaat 240 ggccgcatct ttcgcacgtg tccggcgtgc aaaatatgga tttggcaggatctgctggac 300 agctatgtga atgctttgat aagctactgt cgtgatgcct ccattgattcccttcagtca 360 gagcttgaat ctagccgttt attaatttcc gagaagcagg cacagatttcacgtttggag 420 aaacaattgg agacgctgca gccacttatc tcaaaatata ctgaacaatctcgcagcatt 480 gctcaggcct ccattccatc atcacttttt ttcttggaag cctgcagtctccgacatcag 540 cggcggaggg tgaatgaaaa tcaaggaggg gctaatttca aaagaagctactggagcgat 600 taaagcttcg gttaaaaata agcaagaatc taacacccag agcacaaattccatgagctg 660 gctttttttt gggacaccct ttcatttttc atcaaaaaag ggggggcacccccagtttcc 720 tccaaaaggc tccccctgtc cgacatcata ggtgatgtga ttacccaaaaacaggttgtc 780 ccgcttgctg actcgatgcc aaatttggat tcaatgctgc tcctgttgttttaacaatca 840 atcattttga ctaaaagcat tccccttaaa attgttgtta aatttattgtcaaacttatt 900 accgcaaagt ccgttggcag gtaatccccc ccttttt 937 4 200 PRTSaccharum hybrid cultivar CP72-1210 4 Gly His Tyr Gly Arg Gly Glu HisCys Met Lys Asn Met Ala Ala Ser 1 5 10 15 Tyr Ser Gln Asp Ala Gln LeuAsp Leu Val Leu Pro Asp Ala Pro Val 20 25 30 Asp Ala Ser Ala Ser Arg SerGlu His Ser Ser Gln Leu Ala Ser Ser 35 40 45 Asn Trp Arg Ser Val Ile GlnAsn Ser Pro Pro Asp Leu Leu Cys Gly 50 55 60 Cys Gly Arg Pro Ala Ile ArgArg Thr Ala Glu Thr Ala Lys Asn Asn 65 70 75 80 Gly Arg Ile Phe Arg ThrCys Pro Ala Cys Lys Ile Trp Ile Trp Gln 85 90 95 Asp Leu Leu Asp Ser TyrVal Asn Ala Leu Ile Ser Tyr Cys Arg Asp 100 105 110 Ala Ser Ile Asp SerLeu Gln Ser Glu Leu Glu Ser Ser Arg Leu Leu 115 120 125 Ile Ser Glu LysGln Ala Gln Ile Ser Arg Leu Glu Lys Gln Leu Glu 130 135 140 Thr Leu GlnPro Leu Ile Ser Lys Tyr Thr Glu Gln Ser Arg Ser Ile 145 150 155 160 AlaGln Ala Ser Ile Pro Ser Ser Leu Phe Phe Leu Glu Ala Cys Ser 165 170 175Leu Arg His Gln Arg Arg Arg Val Asn Glu Asn Gln Gly Gly Ala Asn 180 185190 Phe Lys Arg Ser Tyr Trp Ser Asp 195 200 5 449 DNA Saccharum hybridcultivar CP72-1210 5 gaattcggcc attatggccg gggcatgtgc agtgaatgcgccaaggtcct gaggtaccaa 60 accactcggt gccccatctg caggcagcct gttgagcgtctcctcgagat caaagtgagc 120 aacaaatctg aagagcagca gcagacgccc caatcgccgccgctcccagc cccagctctg 180 cagcaggaag aggtgtagcc gtgattaaag tcagttctgagacattatat ggaactagtt 240 tgcggccttc aggcctttcc ctaaaggttt gttctctcatctgagcaacg gggaatgtaa 300 ccggtacttt acctttagcc tatgtaagct tctggcatcgcatggctttg ccgacctctg 360 ctgtacctgc ttatctggag gtcggagacc aagatgccaaggaaagtgtg taccgtatat 420 taaaaaaaaa aaaaaaaaaa aaaaaaaaa 449 6 149 PRTSaccharum hybrid cultivar CP72-1210 6 Glu Phe Gly His Tyr Gly Arg GlyMet Cys Ser Glu Cys Ala Lys Val 1 5 10 15 Leu Arg Tyr Gln Thr Thr ArgCys Pro Ile Cys Arg Gln Pro Val Glu 20 25 30 Arg Leu Leu Glu Ile Lys ValSer Asn Lys Ser Glu Glu Gln Gln Gln 35 40 45 Thr Pro Gln Ser Pro Pro LeuPro Ala Pro Ala Leu Gln Gln Glu Glu 50 55 60 Val Glx Pro Glx Leu Lys SerVal Leu Arg His Tyr Met Glu Leu Val 65 70 75 80 Cys Gly Leu Gln Ala PhePro Glx Arg Phe Val Leu Ser Ser Glu Gln 85 90 95 Arg Gly Met Glx Pro ValLeu Tyr Leu Glx Pro Met Glx Ala Ser Gly 100 105 110 Ile Ala Trp Leu CysArg Pro Leu Leu Tyr Leu Leu Ile Trp Arg Ser 115 120 125 Glu Thr Lys MetPro Arg Lys Val Cys Thr Val Tyr Glx Lys Lys Lys 130 135 140 Lys Lys LysLys Lys 145 7 872 DNA Saccharum hybrid cultivar CP72-1210 7 gaattcggccattatggccg ggaccaggaa ctccaggaat cagatgacaa ctcggggtat 60 agagctcctgaagtgaccat gtccggtcag tattctcaaa agagtgatgt ttacagcttt 120 ggtgtcgtcatgcttgagct actgactgga cagaaagcat ttgacagctc tcgggcaagg 180 tcccagcaatcactagtccg gtgggcttca ccgcagctgc acgacatcga ctcgctagat 240 cagatggttgatccaacctt agaggggctg taccatgcga aatcactctc tcggttcgca 300 gacgcaatcgctctctgtgt ccagcctgaa ccagaattca ggccaccaat gtcggaggtc 360 gtccagtcactggtccgtct tgtgcagcga gcaagcatgg ggacagcact aagcagcgag 420 tggaattcttgccagttcga tgaatctggt gatcacacgc tctaggggaa aatgatgtgt 480 atttcctagagagtctgatg aggaactata gaaggctcac aagtcataga aacttgcagc 540 ttggcattgttgtgagttgt gacggtgtga catgtgccag tgtcaggtga aatgtgactt 600 ttacctatgccattttactg agagtctgct gcaacctgaa gtaggggtga aaagaaagtt 660 ccttctttaaaaatatatat ggttcattcg gacgtgtata tgaatatctt ttgaagacaa 720 tcaactttctgatttcgtct ctgatcgctg tccaaaaatt atcagggaag atgtagcact 780 agtcctgccacagaattagt catctgtata tcctcagaaa tccgaaccat atccaggaaa 840 catcaacagaggacacgtcc acatattcga ac 872 8 290 PRT Saccharum hybrid cultivarCP72-1210 8 Glu Phe Gly His Tyr Gly Arg Asp Gln Glu Leu Gln Glu Ser AspAsp 1 5 10 15 Asn Ser Gly Tyr Arg Ala Pro Glu Val Thr Met Ser Gly GlnTyr Ser 20 25 30 Gln Lys Ser Asp Val Tyr Ser Phe Gly Val Val Met Leu GluLeu Leu 35 40 45 Thr Gly Gln Lys Ala Phe Asp Ser Ser Arg Ala Arg Ser GlnGln Ser 50 55 60 Leu Val Arg Trp Ala Ser Pro Gln Leu His Asp Ile Asp SerLeu Asp 65 70 75 80 Gln Met Val Asp Pro Thr Leu Glu Gly Leu Tyr His AlaLys Ser Leu 85 90 95 Ser Arg Phe Ala Asp Ala Ile Ala Leu Cys Val Gln ProGlu Pro Glu 100 105 110 Phe Arg Pro Pro Met Ser Glu Val Val Gln Ser LeuVal Arg Leu Val 115 120 125 Gln Arg Ala Ser Met Gly Thr Ala Leu Ser SerGlu Trp Asn Ser Cys 130 135 140 Gln Phe Asp Glu Ser Gly Asp His Thr LeuGlx Gly Lys Met Met Cys 145 150 155 160 Ile Ser Glx Arg Val Glx Glx GlyThr Ile Glu Gly Ser Gln Val Ile 165 170 175 Glu Thr Cys Ser Leu Ala LeuLeu Glx Val Val Thr Val Glx His Val 180 185 190 Pro Val Ser Gly Glu MetGlx Leu Leu Pro Met Pro Phe Tyr Glx Glu 195 200 205 Ser Ala Ala Thr GlxSer Arg Gly Glu Lys Lys Val Pro Ser Leu Lys 210 215 220 Ile Tyr Met ValHis Ser Asp Val Tyr Met Asn Ile Phe Glx Arg Gln 225 230 235 240 Ser ThrPhe Glx Phe Arg Leu Glx Ser Leu Ser Lys Asn Tyr Gln Gly 245 250 255 ArgCys Ser Thr Ser Pro Ala Thr Glu Leu Val Ile Cys Ile Ser Ser 260 265 270Glu Ile Arg Thr Ile Ser Arg Lys His Gln Gln Arg Thr Arg Pro His 275 280285 Ile Arg 290 9 1141 DNA Saccharum hybrid cultivar CP72-1210 9gaattcggcc attatggccg gggaccgcag ttcccccgac cacaccgttc cgccgcgcac 60agaggccagc cccgcgccag gagtaagttt gttcttttta acaatatgtc gagggaggag 120aatgtttaca tggccaagct ggctgagcag gccgaaaggt atgaggagat ggttgagtat 180atggagaagg tggctaagac tgtagatgtt gaagagctca ctgtggagga gcgtaacctc 240ctgtctgtcg catacaagaa tgtgattggg gctcgccgtg cttcatggcg cattgtctct 300tccattgaac agaaggagga gtcccgtaag aacgaagagc atgtgaacct tatcaaggaa 360taccgcggga agattgaggc tgaactgagc aacatctgtg atggcatcct gaaactgctt 420gactcccacc tagtgccttc ctctactgct gctgaatcaa aggtcttcta cctcaagatg 480aagggtgact atcacaggta tcttgcggaa tttaagactg gtgctgagag gaaggaatct 540gctgagagca caatggtagc ctacaaggct gctcaggaca ttgctctggc tgagctggca 600cctacacatc cgataaggct tgggcttgct cttaacttct cagtgttcta ttatgagatt 660ctgaactccc cagacaaagc ttgcaacctt gcaaagcagg cgtttgatga agctatctct 720gagttagaca cccttgggga ggagtcatac aaagatagca ctctgatcat gcagctcctg 780agggacaact tgaccctttg gacctctgac ctcacggagg atggtgctga tgagggcaaa 840gaagcctcaa aaggtgatgc tggcgaggga cagtaatctt cggagagggc atgttgttcc 900agcctggttt tagatgctct atgctgtcga agctgtgccg tgccattatt gtagcagatt 960tcctctcccc ctcacttcat ttgcctcata ttagtaggct ggtagtggtc gaattagttc 1020ccattgcttt gtgttgcagc tagttggcac taggtccgtg tggactggta ttgttcccct 1080ggatttgaca agcatgtcct gtggtcgctc tagcgtttta ttgagctttg aagcctcgat 1140 t1141 10 380 PRT Saccharum hybrid cultivar CP72-1210 10 Glu Phe Gly HisTyr Gly Arg Gly Pro Gln Phe Pro Arg Pro His Arg 1 5 10 15 Ser Ala AlaHis Arg Gly Gln Pro Arg Ala Arg Ser Lys Phe Val Leu 20 25 30 Phe Asn AsnMet Ser Arg Glu Glu Asn Val Tyr Met Ala Lys Leu Ala 35 40 45 Glu Gln AlaGlu Arg Tyr Glu Glu Met Val Glu Tyr Met Glu Lys Val 50 55 60 Ala Lys ThrVal Asp Val Glu Glu Leu Thr Val Glu Glu Arg Asn Leu 65 70 75 80 Leu SerVal Ala Tyr Lys Asn Val Ile Gly Ala Arg Arg Ala Ser Trp 85 90 95 Arg IleVal Ser Ser Ile Glu Gln Lys Glu Glu Ser Arg Lys Asn Glu 100 105 110 GluHis Val Asn Leu Ile Lys Glu Tyr Arg Gly Lys Ile Glu Ala Glu 115 120 125Leu Ser Asn Ile Cys Asp Gly Ile Leu Lys Leu Leu Asp Ser His Leu 130 135140 Val Pro Ser Ser Thr Ala Ala Glu Ser Lys Val Phe Tyr Leu Lys Met 145150 155 160 Lys Gly Asp Tyr His Arg Tyr Leu Ala Glu Phe Lys Thr Gly AlaGlu 165 170 175 Arg Lys Glu Ser Ala Glu Ser Thr Met Val Ala Tyr Lys AlaAla Gln 180 185 190 Asp Ile Ala Leu Ala Glu Leu Ala Pro Thr His Pro IleArg Leu Gly 195 200 205 Leu Ala Leu Asn Phe Ser Val Phe Tyr Tyr Glu IleLeu Asn Ser Pro 210 215 220 Asp Lys Ala Cys Asn Leu Ala Lys Gln Ala PheAsp Glu Ala Ile Ser 225 230 235 240 Glu Leu Asp Thr Leu Gly Glu Glu SerTyr Lys Asp Ser Thr Leu Ile 245 250 255 Met Gln Leu Leu Arg Asp Asn LeuThr Leu Trp Thr Ser Asp Leu Thr 260 265 270 Glu Asp Gly Ala Asp Glu GlyLys Glu Ala Ser Lys Gly Asp Ala Gly 275 280 285 Glu Gly Gln Glx Ser SerGlu Arg Ala Cys Cys Ser Ser Leu Val Leu 290 295 300 Asp Ala Leu Cys CysArg Ser Cys Ala Val Pro Leu Leu Glx Gln Ile 305 310 315 320 Ser Ser ProPro His Phe Ile Cys Leu Ile Leu Val Gly Trp Glx Trp 325 330 335 Ser AsnGlx Phe Pro Leu Leu Cys Val Ala Ala Ser Trp His Glx Val 340 345 350 ArgVal Asp Trp Tyr Cys Ser Pro Gly Phe Asp Lys His Val Leu Trp 355 360 365Ser Leu Glx Arg Phe Ile Glu Leu Glx Ser Leu Asp 370 375 380 11 829 DNASaccharum hybrid cultivar CP72-1210 11 gaattcggcc attatggccg ggctgttcaagctggaccgt tatatggtat gggacaccat 60 ggatcttcca ccacaattgc ttatggcggtgcatacttgc catattcttc ctcaactgga 120 caatcgagca ataatcatca agagcatggatttcctgagc ggccagggca gcctgagtgt 180 caatatttta tgaggactgg aggttgcaaatttggaacta tgtgtaaata taaccatcct 240 cgagattgga gcactcctaa gtccaactacatgttcagtc atctctgcct tccacttcgt 300 ccgggtgctc agccttgtgc gtactatgcacaaaatggat attgcagata tggagttgca 360 tgcaaatatg atcacccaat gggtacactaggctacagtt catctgcttt acccctatct 420 gacatgccaa ttgctcccta ccctatcggcttctctgttg ccacgttggc tccatcttca 480 tcttccccag aatatatttc aaccaaagatccatcaatca accaagtagc atcaccagtg 540 cagcacccga acatgttgga acaatcttgccaaaaggggt ttcccttcgg atccattatg 600 cgaactcaac ttctacaagt gtcggcagttcaagcctggg gggcgctgat tttctgactg 660 ggggatgatc cttaacacaa atttctatacttgaacagtt tgaagccttc aaggaataaa 720 aactggggcc ttgaaaaacc gggaggggttcttcccaaat aaaactgtgg tcaacactca 780 tcctgaattg gtttcctatt caaacggaagaggtttagga gtcacattg 829 12 276 PRT Saccharum hybrid cultivar CP72-121012 Glu Phe Gly His Tyr Gly Arg Ala Val Gln Ala Gly Pro Leu Tyr Gly 1 510 15 Met Gly His His Gly Ser Ser Thr Thr Ile Ala Tyr Gly Gly Ala Tyr 2025 30 Leu Pro Tyr Ser Ser Ser Thr Gly Gln Ser Ser Asn Asn His Gln Glu 3540 45 His Gly Phe Pro Glu Arg Pro Gly Gln Pro Glu Cys Gln Tyr Phe Met 5055 60 Arg Thr Gly Gly Cys Lys Phe Gly Thr Met Cys Lys Tyr Asn His Pro 6570 75 80 Arg Asp Trp Ser Thr Pro Lys Ser Asn Tyr Met Phe Ser His Leu Cys85 90 95 Leu Pro Leu Arg Pro Gly Ala Gln Pro Cys Ala Tyr Tyr Ala Gln Asn100 105 110 Gly Tyr Cys Arg Tyr Gly Val Ala Cys Lys Tyr Asp His Pro MetGly 115 120 125 Thr Leu Gly Tyr Ser Ser Ser Ala Leu Pro Leu Ser Asp MetPro Ile 130 135 140 Ala Pro Tyr Pro Ile Gly Phe Ser Val Ala Thr Leu AlaPro Ser Ser 145 150 155 160 Ser Ser Pro Glu Tyr Ile Ser Thr Lys Asp ProSer Ile Asn Gln Val 165 170 175 Ala Ser Pro Val Gln His Pro Asn Met LeuGlu Gln Ser Cys Gln Lys 180 185 190 Gly Phe Pro Phe Gly Ser Ile Met ArgThr Gln Leu Leu Gln Val Ser 195 200 205 Ala Val Gln Ala Trp Gly Ala LeuIle Phe Glx Leu Gly Asp Asp Pro 210 215 220 Glx His Lys Phe Leu Tyr LeuAsn Ser Leu Lys Pro Ser Arg Asn Lys 225 230 235 240 Asn Trp Gly Leu GluLys Pro Gly Gly Val Leu Pro Lys Glx Asn Cys 245 250 255 Gly Gln His SerSer Glx Ile Gly Phe Leu Phe Lys Arg Lys Arg Phe 260 265 270 Arg Ser HisIle 275

What is claimed is:
 1. A method of isolating nucleic acid encoding aplant polypeptide active in PTGS comprising the steps of: i) selecting abait nucleic acid which encodes a bait protein active in PTGS in plantsor suppressive of PTGS in plants; ii) preparing a cDNA prey library froma plant wherein the plant exhibits PTGS; iii) conducting a yeasttwo-hybrid assay with the bait and prey nucleic acids, wherein prey cDNAthat yields a true positive yeast two-hybrid assay result encodes apolypeptide active in PTGS in the plant.
 2. The method of claim 1wherein the bait nucleic acid comprises a sequence selected from thegroup consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 3, SEQ. ID. NO. 5, SEQ.ID. NO. 7, SEQ. ID. NO. 9, and SEQ. ID. NO.
 11. 3. The method of claim 1wherein the plant is a monocot.
 4. The method of claim 3 wherein themonocot is selected from the group consisting of sugarcane, corn,sorghum and rice.
 5. The method of claim 1 further comprising the stepof confirming interaction between the bait protein and the polypeptideactive in PTGS using a farwestern blot assay.
 6. The method of claim 1further comprising the step of confirming interaction between the baitprotein and the polypeptide active in PTGS using a pull down assay. 7.The method of claim 1 further comprising the step of confirminginteraction between the bait protein and the polypeptide active in PTGSusing an in planta assay.
 8. The method of claim 7 wherein the in plantaassay is conducted in embryonic calli of the plant.
 9. An isolatednucleic acid molecule comprising a nucleic acid molecule having at least85% identity with the nucleic acid sequence of SEQ. ID. NO.
 1. 10. Apolypeptide comprising an amino acid having at least 78% identity withthe amino acid sequence of SEQ. ID. NO.
 2. 11. An isolated nucleic acidmolecule comprising a nucleic acid molecule having at least 40% identitywith the nucleic acid sequence of SEQ. ID. NO.
 3. 12. A polypeptidecomprising the amino acid sequence of SEQ. ID. NO.
 4. 13. An isolatednucleic acid molecule comprising a nucleic acid molecule having at least93% identity with the nucleic acid sequence of SEQ. ID. NO.
 5. 14. Apolypeptide comprising an amino acid having at least 95% identity withthe amino acid sequence of SEQ. ID. NO.
 6. 15. An isolated nucleic acidmolecule comprising a nucleic acid molecule having at least 98% identitywith the nucleic acid sequence of SEQ. ID. NO.
 7. 16. A polypeptidecomprising an amino acid having at least 76% identity with the aminoacid sequence of SEQ. ID. NO.
 8. 17. An isolated nucleic acid moleculecomprising a nucleic acid molecule having at least 94% identity with thenucleic acid sequence of SEQ. ID. NO.
 9. 18. A polypeptide comprising anamino acid having at least 97% identity with the amino acid sequence ofSEQ. ID. NO.
 10. 19. An isolated nucleic acid molecule comprising anucleic acid molecule having at least 89% identity with the nucleic acidsequence of SEQ. ID. NO.
 11. 20. A polypeptide comprising an amino acidhaving at least 78% identity with the amino acid sequence of SEQ. ID.NO.
 12. 21. A transgenic plant cell in which PTGS is suppressedcomprising a nucleic acid having a sequence with at least 85% identitywith the nucleic acid sequence of SEQ. ID. NO.
 1. 22. A transgenic plantcell in which PTGS is enhanced comprising a nucleic acid having asequence with at least 40% identity with the nucleic acid sequence ofSEQ. ID. NO.
 3. 23. A transgenic plant cell comprising a nucleic acidhaving a sequence selected from the group consisting of SEQ. ID. NO. 1,SEQ. ID. NO. 3, SEQ. ID. NO. 5, SEQ. ID. NO. 7, SEQ. ID. NO. 9, and SEQ.ID. NO.
 11. 24. A method of increasing viral resistance in a plantcomprising the steps of: i) selecting a protein suppressive of PTGS inthe plant; and ii) transforming the plant with a nucleic acid encodingthe PTGS suppressive protein.
 25. A method of increasing expression of atransgene in a plant comprising the steps of: i) selecting a proteinactive in PTGS in the plant; and ii) transforming the plant with anucleic acid encoding the protein active in PTGS.
 26. A method ofsuppressing expression of a native gene in a plant comprising: i)preparing a vector including a nucleic acid with a sequence of thecoding portion of the gene wherein the nucleic acid, upon transcription,products an mRNA molecule double stranded in the region correspondingthe to the coding portion of the gene; and ii) transforming the plantwith the vector.